Background Problems
The invention concerns problems that are associated with
a) obtaining proper transport of reagents, analytes, washing liquid, buffers etc from an inlet opening to a reaction microcavity in parallel in a plurality of microchannel structures of a microfluidic device,
b) obtaining good stability with respect to wettability of plastic material that are to be in contact with a liquid within the microfluidic format (storage stability and stability towards rewetting once, twice etc,
c) obtaining reduced fouling of reagents, e.g. reagents exhibiting peptide structure, carbohydrate structure, lipid structure, nucleotide structure, nucleic acid structure, hormone structure, steroid structure etc,
d) release of residual monomers, initiators, softeners etc from plastic material,
e) changes of chemical surface characteristics caused by molecular movements and conformational changes in plastics during storage,
f) providing cost effective surface treatment methods that can be used in the large scale manufacturing of microfluidic devices, and
g) provide surfaces that are to be included as inner surfaces in microfluidic devices and which are good substrates for further patterning, attachment of reagents and other functionalizations such hydrophobization, introduction of ionic groups etc.
The miniaturization of the protocols that nowadays are performed in microfluidic systems started during the late eighties. The substrate material of choice was silicon or glass, which inherently have a good wettability (hydrophilicity) for aqueous liquids. In most cases there was no imperative need to separately make the surfaces wettable (hydrophilic).
Later, plastic materials and replication techniques were exploited (e.g. WO 9116966 Amersham Pharmacia Biotech AB; WO 0114116 Åmic AB; WO 0119586 Åmic AB; WO 0021728 Åmic AB). However, plastics inherently have poor wettability and are often hydrophobic. Proper hydrophilization protocols became important.
Downscaling as such will decrease the volume-to-surface ratio, which means a significantly increased risk for undesired consumption/inactivation of reactants, for instance by fouling (non-specific adsorption and/or denaturation).
Fouling and storage stability with respect to hydrophilic/hydrophobic balance were truly overlooked in the initial work when miniturizing. However, these issues are extremely important for the large scale commercialization of microfluidic concepts that utilize devices made in plastic material.
Most likely, the optimal coating of inner surfaces of microfluidic devices will depend on the protocol as such, reagents/reactants and/or liquids to be used, analyte to be assayed, substrate material etc.
For the reasons given above, there is a need for a range of proper treatment protocols for surfaces to be used as inner surfaces in contact with aqueous liquids in microfluidic devices made of plastic material.
Background Publications
Many recent publications in microfluidics present surface treatments in general terms without recognizing the actual problems. See for instance WO 9721090 (Gamera Biosciences) and WO 9958245 (Amersham Pharmacia Biotech AB) where both gas plasma treatment and coating with hydrophilic compounds are mentioned in general terms.
Other recent publications suggest solutions to one or more of the problems discussed above:
WO 0056808 (Amersham Pharmacia Biotech AB) describes gas plasma treatment of plastic surfaces in order to achieve hydrophilic surfaces which have improved storage stability and which are able to withstand repeated contact with aqueous liquid media.
WO 0147637 (Gyros AB) describes coatings for plastic substrates. The coatings expose a non-ionic hydrophilic polymer to a liquid flow and are introduced in order to balance hydrophilicity versus non-specific adsorption. The hydrophilic polymer is typically covalently attached to a base skeleton that in turn binds to a plastic surface. In the case of charged base skeletons, it is suggested to pre-introduce the opposite charges on the naked plastic surface by e.g. gas plasma hydrophilization. Pre-coating of the plastics with a metal layer is also suggested.
U.S. Pat. No. 6,236,083 (Caliper) describe coatings that are resistant towards non-specific adsorption of proteins. The coatings are used for facilitating controlled electro-osmotic flow in the microchannels. Certain variants are similar to those of WO 0147637 (Gyros AB). The supporting substrate is made of an organic polymer, glass or a silica-based polymer.
WO 02075775 (Gyros AB) and WO 0275776 (Gyros AB) describe among others a conductive layer of indium tin oxide in an outlet port for energy desorption ionization mass spectrometry.
WO 0004390 (Zyomyx) presents microfluidic sensor surfaces that have a layered structure comprising an upper organic thin layer resting on a supporting layer. A number of alternative support layers are given, for instance various metal and metal oxide layers including among others indium tin oxide.
The capacity of metal oxide surfaces, such as surfaces of titanium oxide, indium oxide, tin oxide and various mixtures thereof, for forming bonds with functional groups of organic compounds has been utilized for preparing monolayers exposing surfaces of predetermined chemical surface characteristics. The bonds concerned include covalent bonds and non-covalent bonds such as electrostatic interaction. Since metal oxide surfaces often are charged at moderate pH, they often have a pronounced tendency to nonspecifically adsorb compounds that exhibit groups of the opposite charge, such as proteins. The capacity of binding various compounds to this kind of surfaces has been utilized by:
Cohen et al., “Surface modification of inorganic oxide surfaces by graft polymerization” in “Oxide Surfaces”, ed. Wingrave, Marcel Dekker Inc, New York (2001) p. 321-343
Doron et al., “Organization of Au colloids as monolayers films onto ITO glass surfaces: Application of the metal gold coiloid films as base interfaces to construct redox-active monolayers”, Langmuir 11 (1995) 1313-1317
Fang et al., “Soft-lithography-mediated submicrometer patterning of self-assembled monolayer of hemoglobin on ITO surfaces”, Langmuir 16 (2000) 5221-5226
Fang et al., “Anchoring of self-assembled hemoglobin molecules on bare indium-tin oxide surfaces”, Langmuir 17 (2001) 4360-4366,
Hofer et al., “Alkyl phosphate monolayers. Self-assembled from aqueous solution into metal oxide surfaces”, Langmuir 17 2001) 4014-4020
Mandler, “Preparation and characterization of octadecylsilane monolayers on indium-tin oxide (ITO) surfaces”, J. Electroanal. Chem. 500 (2001) 453-460
Oh et al., “Formation of a self-assembled monolayer of diaminododecane and a heteropolyacid monolayer on the ITO-surface”, Langmuir 15 (1999) 4690-4692
Oh et al., “Ionic charge-selective electron transfer at fullerene-multilayered architecture on an indium-tin oxide surface”, Langmuir 16 (2000) 6777-6779
Schwendel et al., “Temperature dependence of the protein resistance of poly- and oligo(ethylene glycol) terminated alkanethiolate monolayers”, Langmuir 17 (2001) 5717-5720
Textor et al., “Structural chemistry of self-assembled monolayers of octadecylphosphoric acid on tantalum oxide surfaces”, Langmuir
Yan et al., “Preparation anf characterization of self-assembled monolayers on indium tin oxide”, Langmuir 16 (2000) 6208-6215
Zotti et al., “Self-assembly of pyrrolyl- and cyclopentadiyldithiophene-4-yl-n-hexyl-ferrocene on ITO electrodes and their anodic coupling to polyconjugated polymer layers”, Langmuir 13 (1997) 2694-2698
Huang et al., Langmuir 17 (2001) 489-498, Huang et al., Langmuir 18 (2001) 220-230, and Kenausis et al., J. Phys. Chem B 104 (2000) 3298-3309 have described sensor surfaces based on a supporting layer of metal oxide coated with polycationic polylysine grafted with poly (ethylene oxide) possibly functionalized with a biologically active molecule. This concept has been suggested for use in sensors of a particular kind of microfluidic apparatus. See poster by Vörös et al., “Controlled biosensor surfaces”. The coating used significantly reduces the nonspecific adsorption of proteins.
Fang et al., supra, have generally suggested that the non-specific adsorption to indium tin oxide surfaces could be used for setting up sensor surfaces, for instance for assays in the biological field.
Yamamoto et al., “Cell-Free Protein Synthesis in PDMS-Glass Hybrid Microreactor in Microfluidic Devices and Systems III, Eds. Mastrangelo et al., Proceedings of SPIE, Vol 4177 (2000) 72-79) have suggested indium tin oxide as heating layer on a glass substrate of a microcavity intended for cell-free protein synthesis.
A capillary device made from plastic material with the inner wall of the capillary channel coated with aluminum oxide is described in AU 21583/99 (Boehringer Mannheim GmbH).